1. Field of the Invention
The present invention relates to increased biosynthesis and isolation of the isoflavonoid phytoalexin compounds, Glyceollins I, II and III, in soy plants grown under stressed conditions, a composition containing said glyceollin(s), a selective estrogen receptor modulator, and a method of treating and preventing breast and ovarian carcinoma while inhibiting selective estrogen receptor modulator-induced uterotrophic effects.
2. Description of the Relevant Art
During endocrine therapy, the antiestrogen tamoxifen has been shown to prevent pre- and postmenopausal breast cancer and to be a beneficial adjuvant therapy for women with estrogen receptor-positive tumors. Ultimately tumors can develop resistance, and in the case of the antiestrogen tamoxifen, tamoxifen can increase the risk of endometrial cancer (Brown, K. 2002. Expert Opin. Drug Saf. 1: 253-267). Consequently, efforts have been made to develop new antiestrogens from both synthetic and natural sources. Many naturally occurring agents, particularly flavonoids, have shown chemopreventive and anticancer potential in a variety of in vitro and in vivo models (Diel et al. 2001. Human Reproduction 16(5): 997-1006; Barnes, S. 1997. Breast Cancer Res. Treat. 46: 169-179; Aronson et al. 1999. Nutr. Cancer 35(2): 130-136). The isoflavone genistein has received much attention over the last few years as a potential anticancer agent due to its wide-range effects on a number of cellular processes. The chemopreventive effects of genistein and other flavonoids have spurred research to discover other naturally occurring flavonoids in soybean and other plants with anticancer activities.
Phytoalexins constitute a chemically heterogeneous group of substances belonging to the various sub-classifications of flavonoids. Phytoalexins are low molecular weight antimicrobial compounds that are synthesized de novo and accumulate in plants as a stress response (Darvill et al. 1984. Ann. Rev. Plant Physiol. 35: 243-275; Paxton, J. D. 1991. In: Emerging Problems Resulting from Microbial Contamination, Sharma et al. (Eds.), CRC Press, Boca Raton, Fla.). The phytoalexins are products of a plant's secondary metabolism, and often accumulate at infection sites at concentrations that inhibit fungal and bacterial growth (Darvill et al., supra; Paxton, J. D., supra). Countless stress factors or physical stimuli induce phytoalexin accumulation including freezing, ultraviolet light exposure and exposure to microorganisms. In addition, compounds referred to as elicitors, either abiotic or biotic, can stimulate the biosynthesis of phytoalexins. Given that the biosynthesis of isoflavonoids, particularly phytoalexins, can be regulated by external factors, the type and amount of hormonally active phytochemicals may vary form source to source. In soybean, several phytoalexins, particularly the glyceollins, are produced under conditions of stress. The glyceollins (mixture of Glyceollin I, II, and III) have been produced in high concentrations using several elicitors and have antimicrobial activity against several plant pathogens (Darvill et al., supra; Graham et al. 1990. Mol. Plant-Microbe Interactions 3(3): 157-166; Daniel et al. 1999. Environ. Health Perspectives 107: 109-114; Rivera-Vargas et al. 1993. Phytochem. 32 (4): 851-857; Rizk et al. 1984. Qual. Plant Foods Human Nutr. 34 (3): 203-210; Bhattacharyya et al. 1986. Physiol and Mol. Pl. Pathol. 29: 227-237; Graham et al. 1991. Mol. Plant-Microbe Interactions 4(1): 60-68).
Studies linking consumption of a phytochemical rich diet to a decreased risk of hormone-dependent tumors such as breast cancer suggest a role for these compounds in prevention or suppression of cancer. In regards to soy rich diets, the two major phytochemicals present are the isoflavones genistein and daidzein, which exist in the aglycone or conjugated form. Studies support the view that isoflavones may have protective effects with regard to estrogen-dependent cancer, particularly breast cancer. A role for isoflavones in the chemoprevention or potential treatment of breast carcinoma has been studied (Constantinou et al. 1998. Am J. Clin. Nutr. 68 (6 Suppl.): 1426S-1430S; Hewitt et al. 2003. Cancer Lett. 192 (2): 133-143). Several animal studies suggest that isoflavones retard cancer development. Newborn female rats treated neonatally with genistein and then exposed to a carcinogen had increased latency and decreased incidence and multiplicity of mammary tumors compared with vehicle-treated animals (Lamartiniere et al. 1995a. Carcinogenesis 16(11): 2833-2840; Lamartiniere et al. 1995b. Proc. Soc. Exp. Biol. Med. 208 (1): 120-123).
However, studies examining dietary genistein effects on estrogen-responsive human breast carcinoma cells implanted into mice suggest the agonist activity of genistein may enhance tumor growth (Barnes, supra). These animal studies using isolated isoflavones have yielded contradictory results as to the effects of these on tumor growth and chemoprevention suggesting a complex role for the effects of flavonoids on cancer cells. The effects of estrogenic compounds such as daidzein or genistein on breast cancer cell proliferation and tumorigenesis may therefore be in part complicated by a complex activity of both agonistic effects on estrogen receptor (ER) function along with tyrosine kinase inhibitory activity at higher doses.
Soybeans grown under stressed conditions were examined to determine if additional unidentified compounds isolated from soybean could further elucidate the role of phytochemicals in breast cancer tumorigenesis and estrogen receptor function. Through these studies, we described the isolation of the glyceollins as a mixture of three isomers (Glyceollin I, II, and III) from stressed soybeans. Functional analysis of these compounds demonstrated that the glyceollins displayed a marked antiestrogenic effect on estrogen receptor signaling; direct binding to the estrogen receptor correlated with a comparable suppression of 17β-estradiol (E2)-induced proliferation in MCF-7 cells (Burow et al. 2001. J. Endocrinol. 86 (4): 1750-1758). Furthermore, in contrast to other isoflavones found in soy, no estrogenic activity was observed with the glyceollins in vitro. From these studies, we hypothesized that the glyceollins represented novel antiestrogenic flavonoids naturally produced by soy that may be relevant to human health.
There is a need to develop new antiestrogens from both synthetic and natural sources. Further, there is a need to reduce the risks of estrogenic effects as has been observed with tamoxifen in uterine tissue. Thus, in view of the antiestrogenic activity of the glyceollins in vitro and its lack of estrogenic activity, the efficacy of the glyceollins as a novel therapy in vivo was determined.